The detection of specified antigens (defined as a substance whose introduction into an animal stimulates the production of antibodies capable of reacting specifically therewith), haptens (a substance requiring additional accessory materials before its introduction into an animal stimulates the production of antibodies specific therefor), analytes and the like substances (hereinafter collectively referred to as ligands) in body fluids such as blood, sputum, urine, and the like has in recent years become of utmost importance in both the research and clinical environment. The detection of ligands, particularly antigens, and antibodies specific therefor can often be related to various disease states and consequently is of extreme usefulness in diagnosis as well as gaining basic understandings concerning the genesis of disease as well as monitoring the effectiveness of therapies therefor.
Consequently, improved methods for detecting ligands in aqueous samples are constantly sought. It is an object of the present invention to improve such immunoassay methods generally by providing improved and novel detectable labels for use therewith.
Immunoassays in general are based upon the immunological reaction between proteins such as antibodies, antibody fragments or even artificially generated peptides (hereinafter collectively referred to as ligand binding partners) and the substance for which they are specific, generally referred to herein as ligands. Immunological reactions are generally characterized by their high specificity and accordingly numerous schemes have been developed in order to take advantage of this characteristic. Typically, such schemes require either purified ligand to compete with the ligand being measured and a labeled and immobilized antibody or ligand binding partner, or multiple immobilized and labeled ligand binding partners reactive between themselves and the ligand. All these methods uniformly rely upon the detectability of the associated label.
Sensitivity of detection in turn is generally related to the type of label employed and the quality and type of equipment available to detect it. For instance, isotopes have traditionally been recognized as providing a high level of sensitivity since present technological capability allows for the detection of a single isotopic atom but, are nonetheless largely disfavored due to the inherent health dangers imposed by radioisotopes. Further, the procedural difficulties necessitated by careful handling and disposal of isotopic reagents have stimulated the search for better labels.
For this reason, two other types of labels have become heavily favored in recent years, to wit fluorescent molecules and enzymes. Enzymes advantageously offer a biological amplification system due to the continuous or persistant chemical activity characteristic of enzymes whereby substrate is transformed into detectable product. Thus, enzymes are, in fact, an indirect label since it is the product of enzyme activity that is detected, not the enzyme itself. Such enzyme or enzyme linked immunosorbant assays (the so-called ELISA systems), however, disadvantageously require extra steps and reagents in order to supply the substrates under conditions suitable for the conversion to, and detection of resultant product.
Fluorescent molecules, although not offering the amplification advantage of enzymes, have found favor due to the simplicity of the equipment required for their detection and associated methods generally. The fluorescent molecules need merely be illuminated at an appropriate frequency and the resultant spectral emissions detected by photodetectors or the like having sensitivity in the appropriate emission spectral region.
Indeed, numerous instruments including for instance the Ortho Spectrum III.TM. (available from the assignee hereof) employ fluorescently labeled antibodies to detect the presence of antigenic markers on cells which are hydrodynamically focused to pass single file, through an illumination zone. Spectrally specific illumination is advantageously provided by a laser and fluorescent emission detected by photodetectors arranged at suitable locations. In fact, numerous fluorescent dyes have been discovered and are commercially available offering fluorescent emissions in a variety of wavelengths.
Despite the availability of an impressive array of fluorescent dyes, there has been a relative lack of molecules which are excited and emit in the long wavelengths, i.e., the red spectrum. Such characteristics are desirable because these wavelengths can be more easily detectable to the exclusion of natural fluorescence from other biomolecules. Such natural fluorescence otherwise creates a background signal often masking the desired signal from the fluorescent label thereby lowering sensitivity.
Further, most fluorescent molecules typically have an emission spectra closer to the ultraviolet range thereby necessitating relatively expensive light sources such as argon lasers and the like. As may be readily appreciated, any added complexity and price of instrumentation reduces their availability to clinical laboratories and hospitals, particularly in light of recent financial pressures imposed by DRG (Diagnosis Related Group) regulations and the like.
Thus, it is an object of the present invention to provide fluorescent labels which can be excited by relatively inexpensive light sources such as helium neon lasers.
Oi et al., in an article entitled "Fluorescent Phycobiliprotein Conjugates For Analyis of Cells and Molecules", The Journal of Cell Biology, 93:981-986 (1982), described the synthesis of a new class of dyes having fluorescence emissions in the orange-red spectral region. These fluorescent molecules, or phycobiliproteins, are derived from various species of algae. Such phycobiliproteins advantageously exhibit comparatively high efficiency at wavelengths in the neighborhood of those desired.
Fluorescence energy transfer is a process sometimes occurring between two molecules, one of which is deemed a donor and the other generally called an acceptor. Typically, the donor molecule is excited by energy of one wavelength (actually a bell-shaped spectrum of wavelengths having a peak or optimum wavelength preferably selected to be at the peak of the available illumination spectrum) and exhibits a fluorescence energy emission curve much like that of the typical fluorophore. The acceptor molecule is preferably chosen to have a suitable excitation wavelength, preferably so that a significant portion of its excitation spectrum falls within the emission wavelength spectrum of the donor molecule. Under optimum conditions the donor peak emission wavelength will be approximately equal to the acceptor peak excitation wavelength. Acceptance by the acceptor molecule of energy by energy transfer mechanisms from the donor molecule results in apparent diminished fluorescence of the donor molecule. The acceptor molecule in turn may be a chromophore, i.e., a molecule exhibiting no innate fluorescence, but will preferably be a fluorescent molecule having its own characteristic fluorescence emission spectra which increases with energy transfer.
Energy transfer in a true fluorescence energy transfer pair is believed to take place by a dipole-dipole energy transfer process the efficiency of which varies with the inverse 6th power of the distance between the donor and acceptor molecules. Thus, changes in fluorescent energy emission may be used to determine proximity relationships between acceptor and donor molecules or molecules to which they are attached. Further, the dipole-dipole energy transfer process, pursuant to Forster's theory, also has an orientation component which affects transfer efficiency. These and other fluorescence energy transfer considerations have been described in a useful reference written by Lubert Stryer entitled "Fluorescence Energy Transfer as a Spectroscopic Ruler", Annuals Review Biochem., 47:819-846 (1978).
This distance sensitive aspect of fluorescence energy transfer has been employed as a labeling technique by Ullman et al. in U.S. Pat. Nos. 4,199,559; 3,996,345; 4,174,384; and 4,261,968. Ullman teaches the employment of a fluorescer-quencher pair of molecules wherein the fluorescer molecule is excited by light of a specified wavelength and fluoresces at a longer wavelength. The presence of a non-fluorescent quencher molecule in close proximity to the fluorescer, however, results in the quenching of this fluorescence thereby contributing to an overall decrease in measurable fluorescence. Accordingly, Ullman's assays employ two different antibodies labeled with either fluorescer or quencher molecules for specific and simultaneous immunological reaction with the ligand to be detected. These methods require the ligand to be polyvalent in nature in order to provide the necessary multiple binding sites for the attachment of multiple antibodies. These methods also require the two members of the fluorescent energy pair to be part of separate reagents.
Alternately, and in the case of monovalent ligands, Ullman teaches the use of a ligand-analog, the substantial proportion of which has the same spatial and polar organization as the ligand to define one or more determinant or epitopic sites capable of competing with the ligand for the binding sites of the receptor or antibody. The ligand-analog differs from the ligand in the absence of an atom or functional group at the site of binding or in having a linking group which has been introduced in place of one or more atoms originally present in the ligand. Typically then, this ligand-analog is labeled with either the fluorescer or quencher molecule and competes with the ligand of the sample for the binding site on an antibody which is labeled with the other molecule of the fluorescer-quencher pair. In this mode, increasing concentrations of ligand may be expected to compete more effectively for the antibody binding sites thereby displacing labeled ligand-analogs and reducing the amount of fluorescent quenching they would otherwise contribute.
Ullman's assays disadvantageously require polyvalency of the ligand or analyte to be detected, or in the alternative, disadvantageously require the production of purified intact ligand-analogs to compete with the ligand. Experience has demonstrated the production of such substances is a generally disadvantageous, difficult, time consuming and expensive proposition. In either case, the molecules of the fluorescer-quencher pair are separate in the absence of immunological reaction.
It is yet another object of the present invention to employ at least two fluorophores, one of which upon excitation exhibits an emission spectrum capable of exciting the second fluorophore which in turn exhibits an emission spectrum measurably detectable and distinguishable from the first emission spectrum and where one of the fluorophores is a phycobiliprotein.
It is still another object of the present invention to simplify the label technology by providing a label having the individual members of a fluorescent energy transfer pair permanently coupled together and where one of the members is a phycobiliprotein.
Recognizing the problems associated with spectrally close excitation. and emission spectra of fluorescent energy transfer pairs, additional ways were sought to increase the spectral difference or Stokes shift between the emission and excitation frequencies in order to increase sensitivity of detection. Glazer and Stryer in Biophysics Journal 43:383 (1983) described the covalent linkage of two phycobiliproteins, namely phycoerythrin and allophycocyanine in order to effect fluorescence energy transfer therebetween. Thus, emission at one frequency resulted in energy transfer and excitation at a larger frequency thereby obtaining a significant Stokes shift.
The Glazer et al. system, however, resulted in a protein-to-protein coupling ratio of about one-to-one of the conjugate thereby yielding little or no increase in efficiency to accompany the useful Stokes shift. Further, phycobiliproteins tend to be large molecules (on the order of 10.sup.5 daltons) and by linking two such large molecules together, an exceedingly large molecule is derived, one which becomes very difficult to employ in a practical manner in various immunoassay applications. The resultant label is so large as to effectively sterically hinder the immunological components, metaphorically describable as the tail wagging the dog.
Consequently, it is still yet another object of the present invention to provide a phycobiliprotein based label having a greater efficiency coupled with a large Stokes shift but not employing multiple phycobiliproteins.
It is yet still another related object to employ fluorescent energy transfer mechanisms to obtain the aforementioned efficiency increase and Stokes shift but to avoid the disadvantages of Glazer and Stryer entailed with one-to-one protein-to-protein coupling ratios.